The relevant concept of sustainability of the present day leads people to think about the treatment of natural resources and particularly in the quanlity and scarcity of water. The serious problems regarding the management of municipal waste in the country, from production, collection and disposal are the challenges facing municipalities and society in general. The increasing use of pharmaceutical drugs most abundant generates a demand for waste that eventually reach the river beds. With advancement of technologies and know you can monitor the…

► There are two purposes in this research, one is the development of the new method which can be used for detection and quantification of triarylmethanes…
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▼ There are two purposes in this research, one is the development of the new method which can be used for detection and quantification of triarylmethanes in fish tissues. The other is that we confirmed validation and utility of triarylmethanes by the method that is according to Commission Decision 2002/657/EC. Homogenized fish tissues were extracted twice with acetonitrile and defatted with n-hexane. HPLC separation was conducted with the RP-18 column. The mobile phases consisted of 0.5 mM ammonium acetate buffer (pH 3.8, adjusted with acetic acid)â ACN (contained 0.1% formic acid) solution. Triarylmethane was determined by LC-ESI-MS/MS in positive mode.
The correlation coefficients of calibration curves with triarylmethane in fish tissues were 0.998 ~ 0.999. The decision limits (CCÎ±) were 0.16 Â± 0.07 Î¼g/kg(MG), 0.15 Â± 0.04 Î¼g/kg(LMG), 0.20 Â± 0.13 Î¼g/kg(CV) and 0.23 Â± 0.12 Î¼g/kg(LCV), and detection capabilities (CCÎ²) were 0.20 Â± 0.09 Î¼g/kg(MG), 0.18 Â± 0.05 Î¼g/kg(LMG), 0.24 Â± 0.16 Î¼gkg-1(CV) and 0.29 Â± 0.15 Î¼g/kg(LCV).
Advisors/Committee Members: Chi-Hsin Hsu (chair), Wei-Hsien Wang (committee member), Jung-Hui Chen (chair).

► The effect of Bacillus Calmette–Guérin (BCG) on the microglia of mice after recovery from infection is incompletely understood. Microglia cells of mice were collected from…
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▼ The effect of Bacillus Calmette–Guérin (BCG) on the microglia of mice after recovery from infection is incompletely understood. Microglia cells of mice were collected from two groups, mice infected with BCG vs. control mice (BCG vs. control). The objective of this study was to compare the proteins and peptides present in the microglia of BCG-challenged mice one-week after treatment relative to control mice and gain insights through differential detection and enrichment analysis. Detection relied on tandem mass spectrometry proteomics. The database search software PEAKS was used to detect peptides and proteins in the treated and control samples. Differential detection was performed using SAS procedures and enrichment classification was completed using PANTHER. The consistently higher number of proteins and peptides (except for one sample) detected in the control samples suggested that BCG impacts the production of proteins. A number of proteins including F-actin-capping protein subunit alpha-2 (P47754), Alpha-enolase (P17182), and myelin basic protein (F7A0B0) were identified in greater abundance in control mice; while chitinase-like protein 3 (O35744), vesicle-associated membrane protein-associated protein A (Q9WV55), and Protein SET (Q9EQU5) were identified only in BCG-treated mice. The differential detection was consistent with similar studies on the neurological activity and inflammation response to BCG-challenged mice. Functional enrichment analysis identified enriched pathways among differentially detected proteins with a differential detection of two (BCG and control mice) associated with inflammation-mediated and microglia activation with Huntington disease (P00029) and Glycolysis (P00024).
Advisors/Committee Members: Rodriguez-Zas, Sandra L. (advisor).

▼ This thesis describes the application of mass
spectrometry (MS) to glycoprotein and oligosaccharide analysis.
Glycosylated proteins are involved in cell-cell and cell-matrix
recognition. Applications of trypsin and proteinase K to hydrolyze
glycoproteins into glycopeptides that are compatible with MS and
MS/MS analysis are investigated. For successful site-specific
analysis of glycans, glycopeptides with short peptide (3-8
residues) are needed. Although trypsin is an important enzyme for
protein identification, proteinase K is superior for site-specific
glycan analysis due to its potential to hydrolyze every
glycoprotein to short glycopeptides. The gas-phase dissociation
pathways, kinetics and energetics of protonated oligosaccharides
are described. The oligosaccharides dissociate via cleavage at the
glycosidic linkages during thermal activation. Using double
resonance experiments, it was established that oligosaccharides
undergo sequential and parallel fragmentation reactions.
Furthermore, dissociation of product ions to secondary ions was
confirmed. Arrhenius activation parameters, Ea and A for protonated
alpha- and beta-linked D-glucopyranose oligosaccharides are
reported.

► The factors affecting peptide fragmentation have been extensively studied in the literature in order to better predict the fragment ion spectra of peptides and proteins.…
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▼ The factors affecting peptide fragmentation have been extensively studied in the literature in order to better predict the fragment ion spectra of peptides and proteins. While there are countless influences to consider, metal cation binding in the gas-phase is particularly interesting. Herein, a comparison of fragmentation patterns of a model peptide series with various charge carriers (H+, Li+, Na+, K+, and Cu+) will assist in determining the location of the preferred binding site of the metal cation and in assessing differences in the fragmentation pattern as a result of this binding site. An interesting observation from these studies reveals abundant x-type fragment ions occurring from the fragmentation of alkali-metal cationized peptides. As these fragment ions have been observed in previous studies by others but not addressed, the factors affecting the formation of these x-type fragment ions are explored.
Additionally, a home-built 193-nm photodissociation tandem time-of-flight mass spectrometer is utilized to study how peptide fragmentation kinetics affect the fragmentation pattern observed. Initially, the fragmentation timescales of various peptides are investigated. Results indicate that longer fragmentation timescales (~10 microseconds) result in an increased number of identified peaks with internal and ammonia loss fragment ions being the most common in comparison to 'prompt' fragmentation timescales (~1 microsecond). Furthermore, b-type fragment ion formation is also favored at longer timescales for the arginine containing peptides investigated.
The fragmentation pattern of several proline containing peptides is examined by collision-induced dissociation and 193-nm photodissociation. Unique fragment ions are observed with each occurring at a proline residue. Few differences are detected between CID and 193-nm photodissociation spectra, indicating that the proline residues direct fragmentation rather than the dissociation method.
In an effort to improve the performance of the photodissociation tandem TOF instrument, the addition of a second source and a dual-stage reflectron are incorporated. The modifications result in improved mass range, signal-to-noise, and increased fragment ion collection efficiencies. High quality mass spectra are acquired across a range of mass-to-charge ratios from ~600 to 1900. Furthermore, the modifications continue to allow investigation of various fragmentation timescales with the addition of an additional timeframe of ~3 microseconds.
Advisors/Committee Members: Russell, David H. (advisor), Hardin, Paul E. (committee member), North, Simon W. (committee member), Schweikert, Emile A. (committee member).

► As a consequence of the widespread use of computers coupled to high-quality printers and different types of papers, forgery, counterfeiting, change of wills, anonymous…
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▼ As a consequence of the widespread use of computers coupled to high-quality printers and different types of papers, forgery, counterfeiting, change of wills, anonymous letter writing and felonious use of the documents have become serious problems. Forensic analysts are always seeking methods that can provide reliable information on whether a specimen collected at the crime scene is linked to the crime or to a source of known origin. Sensitive methods that can provide more detailed characterization of natural or man-made materials or even provide information not previously available to forensic examiners.
Recent advances in rapid solid sampling of materials using laser ablation (LA) coupled to inductively coupled plasma mass spectroscopy (ICP-MS) have led to this analytical method to be regarded as the “gold standard” in the field of elemental analysis for trace level components in solids. Another, emerging, analytical technique that uses the same laser pulse to generate a plasma that can be interrogated with spectroscopy is laser induced break down spectroscopy (LIBS).
The analysis of ink and paper is also possible because of the surface removal effect of laser interactions with the samples. In the present study, printing inks were analyzed using LIBS, LA-ICP-MS and both of them in tandem mode. In the tandem setup, the light generated during the relaxation of the excited species (LIBS) was used to create a spectral signature of the elements, and the mass-to-charge ratio of the ejected particles (ICP-MS) was used to create a mass spectrum.
For a set of 319 printing ink samples, LA-ICP-MS alone provided discrimination greater than 99%. A subset of 43 printing inks, having a very similar elemental profile, was analyzed by tandem LIBS/LA-ICP-MS. The fusion of LIBS and LA-ICP-MS provided additional discrimination through the detection of elements like Ca, Si, Fe, and K by LIBS, that are difficult to detect and confirm using standalone ICP-MS because of the spectral interferences (isobaric and polyatomic) involved. The combination of these two sensors was found to minimize the individual limitations and provide a more complete and representative chemical characterization of printing inks.
Advisors/Committee Members: José R. Almirall, Yong Cai, Jin He, Jaroslava Miksovska, Francisco Fernandez-Lima.

► The aim of this thesis is the analysis and study of fragmentation of the polyether polyols using the soft-ionization technique, the type of ionization employed…
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▼ The aim of this thesis is the analysis and study of fragmentation of the polyether polyols using the soft-ionization technique, the type of ionization employed was MALDI, the fragmentation was mainly achieved using Tandem mass spectrometry (MS/MS) and TOF/TOF instrument of the compounds. I determined that high intensity can be obtained using lithium ions as cationizing agent. I identified the series and the chemical compositions of the polymers.
Advisors/Committee Members: Nagy, Tibor (advisor).

► This study was performed because of the importance of PIBs. I studied PIBs with olefin, chlorine and hydroxyl end groups using a soft ionization technique…
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▼ This study was performed because of the importance of PIBs. I studied PIBs with olefin, chlorine and hydroxyl end groups using a soft ionization technique (DART-MS) using helium gas stream at a temperature of 400 °C, it was important to note that if the temperature is low, the ability of evaporation of the higher molecules to take place is also low. At low temperatures only the only the lower molecules evaporate leaving the higher molecules. Therefore the lower molecules are seen only in the MS spectrum, which will result in lower average molecular weight. In turn if the temperature is increased, the higher molecules can then evaporate but if the temperature is too high this can result in thermal degradation.
In my experiment no sample preparation was necessary – the samples were introduced under ambient conditions manually.
I made measurements in negative and positive ion modes and from the results of the experimental data it can be seen that in negative ion mode I was able to study the intact molecule and determine the number average molecular weight, molecular weight average, polydispersity. In negative ion mode it was impossible to select and study the chlorinated ions because it could not be detected and no fragmentation was observed.
In positive ion mode there was extensive fragmentation with series of intensities which occurred due to in source fragmentation when the polymers where put into the ion source and part of the polymer fragmented. Oxidative side reactions also occurred in the ion source giving rise to series c, normally the polymer spectrum does not contain this series but as the polymer came in contact with the air in the ion source the series was formed- it has a difference of 16( mass of oxygen) from series a. In positive ion mode I was able to select the chlorinated ions and study the fragments, which I observed that the polymer fragments at any of the arms of the initiator moiety, either loosing one arm or both arms.
Advisors/Committee Members: Nagy, Lajos (advisor).

► I studied and carried out my experiments in the Department of Applied Chemistry at the University of Debrecen, Hungary. Using a soft ionization technique (Electrospray…
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▼ I studied and carried out my experiments in the Department of Applied Chemistry at the University of Debrecen, Hungary.
Using a soft ionization technique (Electrospray Ionization), I was able to study the fragmentation and fragmentation pathways of 1,3,5-trisubstituted 2-pyrazoline derivatives in both positive and negative ion modes. In the normal MS mode, we cannot select and study an ion along with its fragments for structural elucidation; we can only gain basic information about the masses of the ions of the sample to be studied. The MS/MS mode on the other hand, makes it possible to gain more knowledge about the structure of the sample being investigated by enabling the fragmentation as well as the separation of the ions.
The MS/MS method was done using a microtof-Q instrument which employed a Quadrupole analyser for the mass selection of the precursor ions and a Time of Flight analyser for the separation of the ions.
Furthermore, I derived a survival yield plot, showing a particular sample (sample 10) which I studied in both its positive and negative ion modes. The survival yield shows the fragmentation stability of the molecules.
Based on the result from the survival yield plot of sample 10, it can be seen that for the deprotonated form of the sample, higher collision energy is needed for the fragmentation of the molecules. Lesser energy is required for fragmentation of the sample in its protonated form.
I also observed that the main types of fragmentation for the 2-pyrazolines are the fragmentation of the substituents and also the fragmentation of the pyrazoline ring.
Advisors/Committee Members: Nagy, Lajos (advisor).

▼ Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.

► The purpose of this research was to develop and validate a method for the detection of low levels of HMTD extracted from building materials using…
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▼ The purpose of this research was to develop and validate a method for the detection of low levels of HMTD extracted from building materials using liquid chromatography with tandem mass spectrometry (LC/MS/MS). Method validation was performed to determine precision, accuracy, sensitivity, and selectivity. For each building material an extraction method was developed, and the percent of HMTD recovered through extraction was determined. Carpet, wood, concrete, and drywall samples were spiked with a known amount of HMTD analytical standard and the HMTD was then periodically extracted from each of the different materials and analyzed using LC/MS/MS. The amount of HMTD recovered from the building materials was then assessed to determine the degradation and recovery time of HMTD and establish which materials would most likely have detectable residues of the explosive in the event of an investigation.

Vermillion, M. L. (2012). Development of a Liquid Chromatography/Tandem Mass Spectrometry (Lc/Ms/Ms) Method for the Analysis of Peroxide Explosive Residues on Building Materials. (Thesis). Oklahoma State University. Retrieved from http://hdl.handle.net/11244/8102

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Vermillion ML. Development of a Liquid Chromatography/Tandem Mass Spectrometry (Lc/Ms/Ms) Method for the Analysis of Peroxide Explosive Residues on Building Materials. [Thesis]. Oklahoma State University; 2012. Available from: http://hdl.handle.net/11244/8102

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

► Mass spectrometry (MS) has emerged over the last two decades as the analytical technique of choice in systems-level protein studies, known as proteomics. Two are…
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▼ Mass spectrometry (MS) has emerged over the last two decades as the analytical technique of choice in systems-level protein studies, known as proteomics. Two are the MS-based approaches generally applied to proteomics: bottom-up (BU), which relies on the proteolytic digestion of proteins into short (~10 amino acids) peptides, and top-down (TD), where proteolysis is omitted, intact proteins are detected and fragmented in gas phase. Both methods present advantages as well as drawbacks. Here, we sought to establish a complete platform to put forward a third MS-based proteomic approach; middle-down (MD). It implies protein digestion as in BU, but aims to generate large peptides which size approaches the one of small intact proteins that are readily analyzed in TD. This novel domain aims to account for the shortcoming of both classical approaches. Until now, the main reasons behind the limited use of MD proteomics have been the lack of easy-to-use cleaving agents capable of producing peptides in the desired 3-15 kDa mass range with high specificity, limitations in MS and allied instrumentation, and the absence of dedicated bioinformatics tools for processing of acquired data. The latter greatly impedes the next milestone in MD proteomics â large-scale analysis. MD can potentially combine the analysis of large portions of proteins carrying set of biologically-relevant modifications â allowing exploring proteins of molecular weight or complexity incompatible with current TD capabilities â with the high-throughput hallmark of BU proteomics. Here, we first in silico evaluated the potential target amino acid residues to produce peptides in the MD mass range within the proteomes of different organisms. This bioinformatics work was followed by an experimental study based on synthetic MD-sized peptides, aimed at determining the optimal MS and tandemMS parameters for large peptide characterization. Next, we pursued two distinct ways of performing MD proteolysis: i) the use of an enzyme and ii) the use of a chemical reagent. We selected the protease Sap9 as a target enzyme for MD, which we fully characterized and successfully applied to the study of a mixture of monoclonal antibodies, where it showed an advantage over traditional BU in terms of reduced introduction of artifacts to the sample, allowing the post-translational modification investigation and unambiguous antibody identification. The chemical cleavage way we addressed via judicious protocol optimization for hydrolysis at the N-terminal side of cysteine with NTCB reagent. We also advanced MD protocols by generation of large (~50 kDa) subunits of monoclonal antibodies through the use of papain and another more specific novel protease, GingisKHAN, combined with new MS signal processing and data analysis capabilities. The developed workflow improved mapping of the connectivity of cysteines involved in inter- and intra-molecular disulfide bridges in antibodies. To summarize, we demonstrated that MD approach to mass spectrometry and proteomics is a powerful, yet…
Advisors/Committee Members: Kussmann, Martin, Tsybin, Yury.

▼ The purpose of this study was to develop a method for identifying homemade explosives (HME), namely ethylene glycol dinitrate (EGDN) and pentaerythritol tetranitrate (PETN), on construction materials to assist in the investigation of clandestine HME laboratories. LC/MS/MS and GC/MS methods were developed, but the GC/MS methods were used for the study. A partial method validation was performed on the GC/MS method to determine the precision and sensitivity. Carpet, concrete, drywall, and wood were spiked with a known amount of each explosive and a time course study was performed. A solid-phase microextraction (SPME) fiber was used to extract the explosives from the construction materials and the fiber was analyzed using the GC/MS. The results were used to determine which construction material would be the best to sample when in a suspected EGDN or PETN clandestine laboratory. The LC/MS/MS method was only useful for PETN, but was not compatible with SPME. The headspace SPME GC/MS method was successful in detecting EGDN and PETN on carpet, concrete, drywall, and wood throughout the time course study. Optimal SPME exposure time was 15 minutes for EGDN and 30 min for PETN using a PDMS SPME fiber at 40 °C. Carpet had the lowest LOD for EGDN but drywall had the lowest LOD for PETN. Based on the time course study, carpet was determined to be the best material to sample for both EGDN and PETN. For EGDN, concrete was the second choice and for PETN, wood was the second choice.

► In quantitative proteomics, many of the LC-MS based approaches employ stable isotopic labelling to provide relative quantitation of the proteome in different cell states. In…
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▼ In quantitative proteomics, many of the LC-MS based
approaches employ stable isotopic labelling to provide relative
quantitation of the proteome in different cell states. In a typical
approach, peptides are first detected and identified by tandemMS
scans prior to quantifying proteins. This provides the researcher
with a large amount of data that are not useful for quantitation.
It is desirable to improve the throughput of current approaches to
make proteomics a more routine experiment with an enhanced capacity
to detect differentially expressed proteins. This thesis reports
the developments towards this goal, including an assessment of the
viability of stable dimethyl labelling for comparative proteomic
measurements and the evaluation of a dynamic algorithm called
Parallel Isotopic Tag Screening (PITS) for the detection of
isotopically labelled peptides for quantitative proteomics without
the use of tandemMS scans.
Advisors/Committee Members: N/A (external-examiner), Dr. Jean Burnell (graduate-coordinator), Dr. Alan A. Doucette (thesis-reader), Dr. Michael A. Quilliam (thesis-reader), Dr. Louis Ramaley (thesis-reader), Dr. Peter D. Wentzell (thesis-supervisor), Not Applicable (ethics-approval), Not Applicable (manuscripts), Not Applicable (copyright-release).

▼ A novel application of Monte Carlo permutation testing that improves the calculation of the peptide match significance levels and detection rate in database search programs is demonstrated. Novel k-permuted decoy databases (where k denotes the type and number of permutations) were evaluated for accurate computation of match significance levels. K-permuted decoy databases were generated by: (a) complete permutations of peptide sequences (Whole), (b) permutation of terminal positions of peptide sequences (End), and (c) permuted peptides that fall within a certain mass tolerance of the tandem mass spectra (Mass-based). The ‘Whole’ and ‘End’ based permutation tests were performed using various indicators of peptide match quality in OMSSA, Crux, and X! Tandem on manually annotated neuropeptide tandem mass spectrometry spectra. Permutation p-values were calculated as the fraction of the permutations in the k-permuted databases with match indicator score as extreme as the original spectra match in the target database. The ‘Whole’ k-permuted decoy databases identified most (up to 100%) neuropeptides, while the ‘End’ k-permuted decoy databases provided better discrimination of the performance between the match indicators. The permutation test based p-values using the hyperscore (X! Tandem), E-value (OMSSA) and Sp score (Crux) match indicators outperformed the other match indicators in the database search programs. The simple indicator of match “the number of matched ions” provided performance comparable to the best match indicators in the OMSSA, X! Tandem, and Crux. Databases of least 105 k-permuted decoy peptides per spectra provided accurate p-values. Overall, the ‘Whole’ and ‘End’ k-permuted decoy databases improved the consensus among the database search programs.
The ability of the k-permuted decoy databases to improve the classifications among correct and incorrect peptide matches was evaluated with ‘Mass-based’ k-permuted decoy database using best match indicator in the OMSSA (i.e., E-value). The evaluation was performed by searching 5806 tryptic tandem mass spectra (671 with annotated peptide entries) against the standard target and combined target-decoy databases. False discovery rate estimates based on the target-decoy approach and known identities of the annotated spectra were used to filter the peptide-spectrum matches. The k-permuted decoy database approach enabled the detection of up to 89% and 87% annotated peptides relative to the OMSSA’s E-value with 82% and 84% identifications in the target database and target-decoy database, respectively. Improvements in performance was due to better performance of the k-permutation decoy database on small and large peptides with less than 13 matched fragment ions and large (insignificant) OMSSA E-values.
Advisors/Committee Members: Rodriguez-Zas, Sandra L. (advisor), Rodriguez-Zas, Sandra L. (Committee Chair), Sweedler, Jonathan V. (committee member), Villamil, Maria B. (committee member), Caetano-Anollés, Gustavo (committee member).

Akhtar MN. Evaluation and assignment of significance levels to peptide identifications from the database search programs using resampling approach. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/73069

University of Bradford

19.
Brown, Emma Louise.
Investigating the use of coca and other psychoactive plants in Pre-Columbian mummies from Chile and Peru : an analytical investigation into the feasibility of testing ancient hair for drug compounds.

► Psychoactive plants have played a significant role in Andean cultures for millennia. Whilst there is evidence of the importance of psychoactive plants in the Andean…
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▼ Psychoactive plants have played a significant role in Andean cultures for millennia. Whilst there is evidence of the importance of psychoactive plants in the Andean archaeological record, none of these are direct proof that these culturally significant plants were used by ancient Andean populations. This project utilised liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the use of psychoactive plants in individuals from cemetery sites in Chile and Peru by analysing hair specimens for a variety of psychoactive compounds. Hair specimens from 46 individuals buried at cemetery sites in the Azapa Valley (northern Chile) belonging to the Cabuza culture (c AD 300 ¿ 1000) indicated around half of these people ingested coca, as evidenced by the detection of BZE in hair specimens. Two individuals from this population tested positive for bufotenine, the main alkaloid in Anadenanthera snuff. There is a specific material culture associated with snuffing. These findings confirm Anadenanthera was consumed in the Azapa Valley. The 11 individuals from Peru came from the necropolis at Puruchuco-Huaquerones in the Rímac valley near Lima. These individuals belonged to the Ichma culture, but would have been under Inca imperial control during the Late Horizon. Although only a small sample, two-thirds tested positive for BZE, suggestive that access to coca was widespread. This project presents a synthesis of the archaeological evidence for the use of various psychoactive plants in Andes. Also presented is the first report of the detection of bufotenine in ancient hair samples and additional data contributing to the understanding of the use of coca in the Andes.

Brown, E. L. (2012). Investigating the use of coca and other psychoactive plants in Pre-Columbian mummies from Chile and Peru : an analytical investigation into the feasibility of testing ancient hair for drug compounds. (Doctoral Dissertation). University of Bradford. Retrieved from http://hdl.handle.net/10454/5785

Chicago Manual of Style (16th Edition):

Brown, Emma Louise. “Investigating the use of coca and other psychoactive plants in Pre-Columbian mummies from Chile and Peru : an analytical investigation into the feasibility of testing ancient hair for drug compounds.” 2012. Doctoral Dissertation, University of Bradford. Accessed September 15, 2019.
http://hdl.handle.net/10454/5785.

MLA Handbook (7th Edition):

Brown, Emma Louise. “Investigating the use of coca and other psychoactive plants in Pre-Columbian mummies from Chile and Peru : an analytical investigation into the feasibility of testing ancient hair for drug compounds.” 2012. Web. 15 Sep 2019.

Vancouver:

Brown EL. Investigating the use of coca and other psychoactive plants in Pre-Columbian mummies from Chile and Peru : an analytical investigation into the feasibility of testing ancient hair for drug compounds. [Internet] [Doctoral dissertation]. University of Bradford; 2012. [cited 2019 Sep 15].
Available from: http://hdl.handle.net/10454/5785.

Council of Science Editors:

Brown EL. Investigating the use of coca and other psychoactive plants in Pre-Columbian mummies from Chile and Peru : an analytical investigation into the feasibility of testing ancient hair for drug compounds. [Doctoral Dissertation]. University of Bradford; 2012. Available from: http://hdl.handle.net/10454/5785

► Nordihydroguaiaretic acid (NDGA), is a naturally-occurring lignan isolated from the creosote bush (Larrea tridentata). The aqueous extract of this shrub, commonly referred to as Chaparral…
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▼ Nordihydroguaiaretic acid (NDGA), is a naturally-occurring lignan isolated from the creosote bush (Larrea tridentata). The aqueous extract of this shrub, commonly referred to as Chaparral tea, was listed in the American pharmacopeia as an ethnobotanical used to treat tuberculosis, arthritis and cancer. Other documented traditional applications of creosote bush extract include treatment for infertility, rheumatism, arthritis, diabetes, gallbladder and kidney stones, pain and inflammation among many others. In spite of the numerous pharmacological properties, NDGA use has been associated with toxicities including hepatotoxicity in humans. Previous studies in our group showed that oxidative cyclization of NDGA (a di-catechol) at physiological pH forms a dibenzocyclooctadiene that may have therapeutic benefits whilst oxidation to ortho-quinone likely mediates toxicological properties.
In order to investigate the structural features responsible for pharmacological and toxicological properties, a series of NDGA analogues were designed, synthesized and characterized for the purpose of studying their oxidative metabolism. Literature procedures were modified to successfully prepare seven lignan analogues via multi-step synthesis. In our effort to understand the mechanisms of NDGA intramolecular cyclization, the prepared analogues were incubated under previously established conditions where NDGA autoxidized to yield the dibenzocyclooctadiene derivative. We also evaluated the stability of the analogues under the conditions of this study. Furthermore, we evaluated bioactivation potential of the prepared analogues with a goal of eliminating reactive metabolite liability through rational structural modification. We incubated NDGA and its analogues in rat liver microsomes (RLM) in the presence of glutathione as a nucleophilic trapping agent. Standards for comparison were generated by performing glutathione trapping experiments with chemical and enzyme oxidation systems. The potential of the dibenzocyclooctadiene lignan 2 derived from NDGA under physiological conditions to contribute to toxicological properties via reactive metabolite formation was also evaluated. Glutathione conjugates were detected by electrospray ionization-mass spectrometry (ESI-MS) scanning for neutral loss (NL) 129 Da or 307 Da in positive ion mode or precursor ion (PI) scanning for 272 Da in negative ion mode and further characterized by liquid chromatography–tandem mass spectrometry (LC–MS/MS) or in a single LC-MS run using multiple reactions monitoring (MRM) as a survey scan to trigger acquisition of enhanced product ion (EPI) data.
We determined that NDGA autoxidation at pH 7.4 is dependent on substituents and/or substitution pattern on the two aromatic rings. In particular, spontaneous intramolecular cyclization to a dibenzocyclooctadiene required a di-catechol lignan, raising the possibility that o-Q formation may not be necessary for cyclization to occur. Cyclization was significantly inhibited in the presence of excess GSH which supports the…
Advisors/Committee Members: Krol, Edward S., Remillard, Fred A., Palmer, David R., Bandy, Brian, Gravel, Michel.

▼ Chronic, noncommunicable diseases such as chronic fatigue syndrome (also known as
myalgic encephalomyelitis) are rapidly becoming a worldwide epidemic that profoundly
affects public health and productivity. Chronic fatigue syndrome (CFS) is characterised by
severe and debilitating fatigue and although its etiology is still unknown, recent studies have
found considerable evidence that mitochondrial dysfunction and oxidative stress might be
responsible for the underlying energy deficit in these patients. Adenine and pyridine
nucleotides could be used as potential biomarkers for energy related disorders such as
chronic fatigue syndrome because of their various functions in the energy and redox
pathways.
The first part of this study focussed on developing a liquid chromatography electrosprayionisation
tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of
these nucleotides in blood samples. Due to the instability of nucleotides in biological
matrices it was also necessary to find a suitable extraction method that would be able to stop
enzymatic activity via protein precipitation. Out of the four extraction methods investigated
during this study, deproteinisation of whole blood samples with perchloric acid produced the
highest nucleotide abundances. Although nucleotide standards were found to be stable in
perchloric acid, nucleotide levels in blood samples were not stabilised by addition of
perchloric acid.
The second part of this study consisted of measuring the nucleotide levels in blood samples
of controls and possible CFS patients in order to test the proof of concept of the new LCESI-
MS/MS method. Despite changes in the nucleotide levels due to perchloric acid and
problems with nucleotide instability, it was still possible to distinguish between the two
groups based on the results obtained with the new LC-ESI-MS/MS method.
The newly developed LC-ESI-MS/MS method proved to be reliable and adequate for
nucleotide quantification in whole blood samples, thus the aim of this study was achieved.

▼ Tobacco harm reduction involves strategies designed to reduce the harms associated with tobacco use, for example by cutting down cigarette consumption and thereby reducing exposure to the risks associated with smoking. In addition to potentially reducing the harms of continued smoking, there is evidence that some harm reduction strategies are positively associated with quitting smoking entirely. A variety of interventions exist that can be used to aid both cessation and smoking reduction. However, there are limitations with using current treatments, with most attempts to change smoking behaviour ultimately ending with relapse. Numerous limitations of current treatments have been noted in the past, including that existing nicotine replacement products may not provide sufficient nicotine, and / or that the time course of nicotine delivery may not be optimal for promoting reduction. The broad objective of this thesis is to understand how smoking reduction influences smoking behaviours and cessation outcomes as well as smoking-related harm exposure.
To achieve this aim, firstly, knowing the content of nicotine in supplementary products can help with interpreting the outcomes of clinical trials and with improving product design. Vaporised nicotine products (VNPs) – commonly known as electronic cigarettes – are increasingly being used by smokers to reduce their smoking, and there is some evidence that they can be effective cessation aids. A key issue with testing the effectiveness of these products, however, as been quantifying the variability in the amount of nicotine contained in products. As such, an assay was developed and applied to determine the nicotine content in one brand of VNPs which consisted of a fibrous pad in the cartridge. In addition to finding substantial variation between product batches, we also found that the measured nicotine content in the cartridge was lower than the stated content on the product label. The accurate determination of the quantity of nicotine in VNPs has important implications for both consumer safety and the further study of these devices.
Secondly, smoking reduction is not only a reduction in the number of smoked cigarettes, but also an expected reduction in exposure of smoking-related harm. Biological markers can be used to indicate the intake of nicotine and smoking-related harm. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is a tobacco specific carcinogen. As such, NNAL is considered a biological marker associated with tobacco-related harm. To measure the levels of NNAL in smokers’ urine, solid phase extraction (SPE) assay has been used in previously reported studies. However, the sample preparation procedures required with these SPE assays are complicated and time consuming, limiting their use. A simple and efficient liquid-liquid extraction assay combined with ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was designed in this study, and applied to the…

Venlafaxine elicits a small number of adverse effects, so it is one of the most frequently prescribed drugs to treat major depression, generalized anxiety, and social anxiety disorders in adults. In this study, venlafaxine (VEN), O-desmethylvenlafaxine (ODV), and N-desmethylvenlafaxine (NDV) were pre-concentrated with the aid of miniaturized SPE based on MIPs as extraction phase. MIPs are synthetic polymers with cavities specifically designed to hold a target molecule or structurally similar compounds. The molecularly imprinted…

Selenium is an essential micronutrient for many living organisms including man. Its role is related to selenoproteins which contain genetically encoded selenocysteine. There are 25 selenoproteins encoded in the human genome. Their function, expression kinetics and hierarchy have been a topic of intense research in life sciences. There is another type of proteins which contain selenium inserted non-specifically by partly replacing sulphur in methionine and, potentially, cysteine. They are of interest in nutrition science as source of bio-available selenium in natural and supplemented foods. The goal of this Ph.D. was the development of methodologies for the analysis of selenium-containing proteins on the entire proteome scale. A novel procedure was developed for their global detection in 2D electrophoretic gels par laser ablation inductively coupled plasma mass spectrometry (ICP MS) imaging permitting to avoid the use of the radioactive 75Se. The other developments included (i) a robust capillary HPLC – ICP MS coupling allowing the…

▼ Synthetic polymers are the products of humans’ attempts to imitate nature’s gigantic molecular chain architectures. The extended variety of building blocks and reaction mechanisms resulted in a plethora of different polymeric architectures. The biggest challenge for polymer chemists is to develop an understanding of the relation between the chemical structure of polymers and their physicochemical and mechanical properties. Mass spectrometry (MS) can provide detailed information about the elemental composition, monomer unit and end-group structure of polymeric systems. However, it also has its limitations. The analysis of high molar mass and/or disperse and/or structurally complicated synthetic polymers remains a big challenge. This thesis tries to address this challenge by controlling the charge state and energy of synthetic polymers during MS. Chapter 2 gives a general introduction to polymer analysis and MS. Besides this introduction, Chapter 2 presents some examples of current practical state-of-the-art MS, and liquid chromatography coupled to MS, methods for the analysis of synthetic polymers. Chapter 3 provides an example of the current performance of LC coupled to tandemMS (MS/MS) in the analysis of structurally complicated polymers, such as vegetable oil ethoxylates. Chapter 4 presents the method development to achieve an accurate and reproducible control of the applied excitation energy in a quadrupole ion trap. The method is checked by studying the required excitation energy for fragmentation of poly(ethylene glycol)s as a function of their size. This dependence is shown to be linear and in agreement with other MS instruments where the applied excitation energy can be controlled in a more accurate way. In Chapter 5, the methodology for accurate and reproducible control of the applied excitation energy is used to discriminate between different polymer classes. It is shown that discrimination is achieved by determining a “characteristic” parameter (i.e., the characteristic collision voltage (CCV)), which is related to the polymer’s structure and expresses the stability of the respective polymer ion upon excitation. The determination of the CCV is then used in the analysis of a mixture of a structurally complicated copolymer system and various, nominally isobaric, homopolymers, which cannot be discriminated by conventional MS and MS/MS methods. Chapters 6, 7 and 8 present the study of non-covalent complex ions of high molar mass synthetic polymers and molecules containing ammonium functionalities. These complex ions appear to have a preference for low charge states. Chapter 6 and 7 present the investigations of the parameters that influence the formation of these low charge state adducts ions. Chapter 8 presents the behavior of these non-covalent complex ions upon activation at both low and relatively high collision regimes. The amount and type of fragments ions is strongly influenced by the structure of the ammonium ion. The results show that MS/MS of these non-covalent complex ions can be used as a source of…
Advisors/Committee Members: Heeren, R.M.A., Brink, O.F. van den.

▼ Biosynthetic pathway engineering is rapidly growing by
rationally harnessing the enzymatic potential of microbial systems.
While marine cyanobacterial genus and a few gram positive bacteria
(such as Saccharopolyspora erythraea) have offered an extensive
array of promising biocatalysts with unique modular functions as
polyketide synthases (PKS), non-ribosomal peptide synthetases
(NPRS) or their hybrids, the biosynthetic potential of soil
cyanobacteria and marine Nocardiopsis genus largely remain
unexplored. Herein we study a marine actinomycete, Nocardiopsis sp.
CMB-M0232 isolate as a model organism through an integrated
approach involving genome sequencing, metabolic engineering,
tandem-MS and asymmetric synthesis to reconstruct multiple
biosynthetic pathways leading to PKS, NRPS, alkaloids and their
hybrids as potential candidates for clinically relevant drug
development. Chapter 1 and Appendix 2 introduce the reader to the
significance of marine actinomycetes in the context of drug
discovery and development. Chapters 2, 3 and 4 describe multiple
recent success stories on the mechanistic investigations of novel
biocatalytic systems discovered through this integrated approach
applied to Nocardiopsis, for the first time. Specifically, Chapter
2 describes the metabolic pathway to nocardioazines that are
candidates for anti-tumor drug development through their
participation as inhibitors of p-glycoprotein-mediated efflux.
Their biosynthetic pathway is dissected. Chapter 3 builds on the
noz gene cluster and NozA as a biocatalyst. Chapter 4 describes the
nsn-coded modular megasynthase-based biosynthetic machinery for the
assembly of the nocardiopsins. The nocardiopsins are
polyketide-non-ribosomal peptide hybrid natural products that are
structurally homologous to the rapamycins but are indeed distinct
at select positions. Therefore, the nocardiopsin biosynthetic
pathway is not only interesting and significant for the potential
of creating new mTOR pathway analogs for their biological
elucidation. But fundamentally is interesting for biosynthesis of
heterocyclic rings such as tetrahydrofurans, pipecolates and
proline-containing macrolide architectures. Chapter 5 describes the
experimental details, and the corresponding NMR spectra are
provided in Appendix 1. Overall, several novel lines of
investigations are underway and the data gathered and presented
herein constitutes the body of work for three distinct
publications.
Advisors/Committee Members: Viswanathan, Rajesh (Advisor).

Food suppliers are suffering from an intensified price competition with each other, and as a result of this economic pressure, many of them reduce the production cost by decreasing the product quality. Taking into account the number of incidents observed in the last…

► Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions.…
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▼ Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids influence their biological activity, there is a need for comprehensive methods for quantitation of as many individual subspecies as possible. This dissertation describes methods that have been developed and validated for the extraction, liquid chromatographic separation, identification and quantitation of sphingolipids by electrospray ionization (ESI), tandem mass spectrometry (MS/MS) using an internal standard cocktail developed by the LIPID MAPS Consortium. The compounds that can be readily analyzed are sphingoid bases and sphingoid base 1-phosphates, as well as more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, and mono- and di-hexosylceramides. For broader utility, the methods have been optimized for two categories of tandem mass spectrometers. With minor modifications, these methods can be applied to the analysis of isomers such as glucosylceramide and galactosylceramide, and with the availability of additional internal standards, more complex species such as sulfatides can also be quantified. Using these methods 46 species of these compounds have been quantified in RAW264.7 cells, a macrophage cell line. Quantitation of individual sphingolipid metabolites is possible using liquid chromatography, tandem mass spectrometry, and stable isotope labeling with [13C]palmitic acid can be used to differentiate between metabolites produced by de novo synthesis versus turnover. This approach is more accurate when one knows the isotope enrichment of the precursor pool (in this case, [13C]-palmitoyl-CoA); therefore this dissertation describes methods to analyze both the various isotopic forms of palmitoyl-CoA and sphingolipids through sphingomyelins and monohexosylceramides using two cell models, HEK293 cells and RAW264.7 cells treated with Kdo2-Lipid A. The sphingolipid analysis was simplified by the fragmentation of most of the metabolites to backbone product ions. For example the presence of the isotopic label in the long chain base, N-acyl linked fatty acid, or both was determined via, m/z 264 for [12C]sphingosine (d18:1) and m/z 280 for [13C]sphingosine (m+16, d18:1), versus the m/z of the isotopically labeled precursor, (m+16 versus m+32).
Advisors/Committee Members: Merrill, Alfred H. Jr. (Committee Chair), Doyle, Donald (Committee Member), Fernandez, Facundo (Committee Member), Lieberman, Raquel (Committee Member), Sullards, M. Cameron (Committee Member), Wang, May (Committee Member).